CN108030793A - A kind of complex microorganism preparations for preventing aspermia or oligospermia and preparation method thereof - Google Patents

A kind of complex microorganism preparations for preventing aspermia or oligospermia and preparation method thereof Download PDF

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CN108030793A
CN108030793A CN201711496530.XA CN201711496530A CN108030793A CN 108030793 A CN108030793 A CN 108030793A CN 201711496530 A CN201711496530 A CN 201711496530A CN 108030793 A CN108030793 A CN 108030793A
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蒋常德
蒋常银
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Foshan Yanhui Biotechnology Co ltd
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Abstract

Complex microorganism preparations the invention discloses prevention aspermia or oligospermia and preparation method thereof；The raw material for preparing the microorganism formulation is：Pink pod sulphur fermented liquid, coronoid process dissipate capsule bacterium seed, bacillus amyloliquefaciens zymotic fluid, brown sugar, soybean separation protein white powder, honey, sodium chloride, xylo-oligosaccharide, deionized water etc.；The complex microorganism preparations of the present invention contain substantial amounts of microbial polysaccharide material and active probiotic, lead to essence with soothing the liver regulating the qi flowing in the channels, kidney tonifying, adjust the effect such as enteron aisle, clinical symptoms and sign can be obviously improved, Sperm Parameters and Endocrine Levels are improved, improves infertility and disease, the course of disease can be shortened, curative effect is improved, market prospects will be very extensive.

Description

A kind of complex microorganism preparations for preventing aspermia or oligospermia and preparation method thereof
Technical field
The invention belongs to microorganism medicine and food beverage technology field, is specifically related to prevent answering for aspermia or oligospermia to a kind of Close microorganism formulation and preparation method thereof.
Background technology
Aspermia or oligospermia (oligospermatism) refers to that the sperm number in sperm has fecundity male's less than normal A kind of illness, the sperm concentration of the World Health Organization (World Health Organization, WHO) regulation male is in every milli It is oligospermatism less than 2,000 ten thousand to rise in sperm, and fertility aspect will have a significant impact.Counted according to WHO, in past 50 years, The sperm concentration of normal men averagely reduces 40%- 50%, and sperm quantity is declined with annual 0. 25% speed.Every seven couples of Mr. and Mrs 1 couple of infertility person is just there are about, worldwide, infertile patients are up to 50,000,000-8,000 ten thousand, and every year with 2,000,000 pairs of infertility The speed increase of Mr. and Mrs.10% or so of married couple is accounted in Chinese infertile couples, wherein because of male fecundity defect institute Infertility person is caused to be no less than the 50% of infertile couples.Male sterility patient is in the trend that increases year by year in recent years in China, male genetic Health is troubling, and the male sterility using oligospermatism and low sperm activity disease as clinical manifestation is brought to the life of people Greatly challenge, become one of common disease for influencing people's life.
It is generally believed that in the past 50 years, hormone-content is excessive in environmental pollution, food, venereal disease is spread, takes drugs, indulges in excessive drinking, excessively Smoking, life stress are excessive, some medicines such as amcinonide, nervous system medicine, cardiovascular system drug and chemotherapeutics long-term Using etc. biology, physics, chemistry and psychological factor cause genital orgnas,male kingfisher ball damage, be to cause mankind spermatozoon quality sum number The main reason for amount declines.The traditional Chinese medical science thinks that the aspermia or oligospermia cause of disease is mainly that congenital ticket assigns deficiency, intemperance in sexual life, prolonged illness overstrain, diet Do not save, the turbid extravasated blood of phlegm, excessive poison infect, feelings will is unsuccessful etc. causes few essence infertility.
Doctor trained in Western medicine is limited for the existing treatment means of aspermia or oligospermia, there is auxiliary procreation technology such as inseminatio externalis and embryo transfer (monosperm microinjection (ICI) etc. in IVF-ET, archiblast, drug therapy are generally carried using clomiphene, arginine, emerald green ball ketone High sperm concentration, but above-mentioned treatment means are low with success rate, the drawbacks such as drug side-effect is larger.Therefore need to look for it is new safely, have The medicine of effect is very necessary.
The complex microorganism preparations of the present invention obtain important breakthrough in the few smart sterility for the treatment of, solve traditional treatment medicine Thing weak curative effect and the big problem of toxic side effect, can be used as medicine and healthcare applications, and social effect is huge, spy's application the invention Patent.
The content of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of composite microorganism viable bacteria content is high, activity is high, Stability is high, action effect is fast, without side-effects, holding time length, has the complex microorganism preparations of obvious prevention aspermia or oligospermia, The raw material for preparing the microorganism formulation is by weight：Pink 20-40 parts of pod sulphur fermented liquid, coronoid process dissipate capsule bacterium seed 5-10 Part, 20-40 parts of bacillus amyloliquefaciens zymotic fluid, 3-6 parts of brown sugar, 2-4 parts of soybean separation protein white powder, 1-2 parts of honey, sodium chloride 0.2-0.4 parts, 0.5-2 parts of xylo-oligosaccharide, 80 parts of deionized water.
To achieve these goals, the preparation method of the complex microorganism preparations of present invention prevention aspermia or oligospermia, including it is as follows Step：
3-6 parts of brown sugar, 2-4 parts of soybean separation protein white powder, 1-2 parts of honey, 0.2-0.4 parts of sodium chloride are added in fermentation tank, 0.5-2 parts of xylo-oligosaccharide, 80 parts of deionized water, keep the temperature 50 minutes at 105 DEG C again after mixing, are then cooled to 30 DEG C；
Added in (1) coolant that step is produced：The 5-10 parts of 28-30 DEG C of ventilation cultures of coronoid process dissipate capsule bacterium seed, liquid per minute Gas is 1 than throughput:1, open stirring 150r/min culture 72-96 it is small when, treat that thalline content reaches 40g/L, exocellular polysaccharide content Reach 1 g/L, i.e., be transferred to pink 20-40 parts of pod sulphur fermented liquid to fermentation tank, continue cultivate 48-72 it is small when, detect its it is residual also Raw sugar is less than 1g/L；It can terminate second stage fermentation；
20-40 parts of bacillus amyloliquefaciens zymotic fluid is added in (2) zymotic fluid that step is cultivated, it is not low to detect its total viable bacteria In 3 × 109CFU/ml, spore content are not less than 2 × 109CFU/ml, you can filling, packed products.
Wherein, the preparation method of the pink pod sulphur fermented liquid, includes the following steps：
, semi-solid seed activation culture：Pink pod sulphur strain is punctured in semi-solid purple sulphur photosynthetic bacteria culture medium, 25- 35 DEG C of illumination cultivations 7-10 days, bacterium line to be punctured redden and grow lawn, you can for the strain of activation
, seed culture：The strain of activation is seeded in seed fluid nutrient mediums of saccharomycete, 25-35 DEG C of temperature, intensity of illumination is： 1000-3000lux, illumination Anaerobic culturel 7-10 days detect OD650 >=1.2 of seed, and the cfu/ml of viable count >=600,000,000 is kind Sub- nutrient solution；
, fermented and cultured：By seed culture fluid and pink pod sulphur bacteria fermentation culture medium with 1：5 inoculum concentration inoculation, is trained in illumination Supporting Anaerobic culturel 7-10 days, 25-35 DEG C of cultivation temperature, intensity of illumination in tank is：1000-4000lux, mixing speed for 50 turns/ Minute, its OD650 >=4 to be detected, the cfu/ml of viable count >=1,000,000,000, polyoses content reaches 5 g/L, is pink pod sulphur bacterium fermentation Liquid；
Wherein, the purple sulphur photosynthetic bacteria culture medium of the semisolid is：Ammonium chloride 0.6g/L, potassium dihydrogen phosphate 0.6g/L, two water Calcium chloride 0.08g/L, magnesium chloride 0.3g/L, fructose 2g/L, 2 g/L of sodium acetate, natrium malicum 1g/L, nine water vulcanized sodium 0.2g/ L, agar 10g/L, 121 DEG C sterilize 15 minutes, with acetic acid tune PH to 7.0-7.2, wherein nine water vulcanized sodium are first configured to 0.1g/mL Individually sterilizing；
Wherein, the seed fluid nutrient mediums of saccharomycete is：Ammonium chloride 0.6g/L, potassium dihydrogen phosphate 0.8g/L, calcium chloride dihydrate 0.05g/L, magnesium chloride 0.3g/L, fructose 2g/L, 2 g/L of sodium acetate, glycerine 0.5g/L, nine water vulcanized sodium 0.2g/L, 121 DEG C go out Bacterium 15 minutes, with acetic acid tune PH to 7.0-7.2, individually sterilizes wherein nine water vulcanized sodium are first configured to 0.1g/mL；
Wherein, the pink pod sulphur bacteria fermentation culture medium is：Soybean protein isolate 20 g/L, ammonium chloride 0.6g/L, di(2-ethylhexyl)phosphate Hydrogen potassium 0.6g/L, calcium chloride dihydrate 0.08g/L, magnesium chloride 0.2g/L, fructose 20g/L, sodium acetate 2 g/L, honey 10g/L, sulphur Sodium thiosulfate 1g/L, 121 DEG C sterilize 15 minutes, with acetic acid tune PH to 7.0-7.2；
Wherein, pink pod sulphur bacterium is deposited in China General Microbiological Bacterial Strains Managing center by the present inventor, and numbering is CGMCC10344.Preservation date is on January 12nd, 2015.
Wherein, the preparation method of the bacillus amyloliquefaciens zymotic fluid, includes the following steps：First, strain is taken out to protect Pipe is hidden, tablet is drawn with LB solid mediums and recovers, when 33 DEG C of cultures 48 are small.50 milli of picking single bacterium colony inoculation under tablet Rise in LB culture mediums, when 33 DEG C of shake cultures 24 are small in the incubator.Seed uses 5% inoculum concentration, and it is 2L to be seeded to liquid amount In the big triangular flasks of 5L of LB culture mediums, when 33 DEG C of shake culture 20-28 are small, its bacterial concentration is detected, viable bacteria content is more than 20 Hundred million/milliliter, you can as bacillus amyloliquefaciens liquid spawn；
2nd, using the bacillus amyloliquefaciens liquid spawn of step 1 culture as seed, 600L fermentation mediums is seeded to, are inoculated with Measure as 10%, open stirring 120r/min, minute ventilation volume liquid-gas ratio 1：When 0.8,33 DEG C of culture 24-36 is small, treat spore content not Less than 10,000,000,000/milliliter, you can terminate fermentation；
Wherein, the LB culture mediums：Peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, in solid medium Add agar 2%；
Wherein, the fermentation medium：Soybean protein isolate 40 g/L, glucose 10g/L, 20 g/L of soluble starch, albumen 8 g/L of peptone, 0.3 g/L of magnesium sulfate, 0.2 g/L of manganese sulfate, 0.5 g/L of sodium dihydrogen phosphate, 2.3 g/L of disodium hydrogen phosphate, pH7.2；The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes；
Wherein, the bacillus amyloliquefaciens are bacillus amyloliquefaciens（Bacillus amyloliquefaciens ） YHSH-58 is by the patentee in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese microorganism strain preservation conservator Meeting common micro-organisms center preservation, deposit number is that CGMCC NO.13664 preservation dates are on 2 16th, 2017.
Wherein, the coronoid process dissipate capsule bacterium seed liquor is prepared as follows：Coronoid process dissipate capsule bacterium culture presevation pipe is taken out, is used PDA solid mediums draw tablet and recover, and 28 DEG C are cultivated 5 days, and the streak inoculation of picking single bacterium colony is equipped with 150 millis under tablet In the eggplant bottle for rising PDA solid mediums, cultivate 5-7 days for 28 DEG C in the incubator, treat that lawn covers with eggplant bottle, produce a large amount of spores The i.e. 500 milliliters available sterile saline elution of son, adjusting spore concentration is 100,000,000 cfu/ml, is coronoid process dissipate capsule bacterium seed Liquid；
Wherein, coronoid process dissipate capsule bacterium strain is available from China General Microbiological culture presevation administrative center, numbering CGMCC 3.7928。
The pink pod sulphur bacterium contained in the present invention, the present inventor, which has it, compares thin research and experiment, starts main The degradation capability and ammonia nitrogen removal ability of its superpower hydrogen sulfide are studied emphatically, therefore main application is focused on as microbial bacteria The fields such as fertilizer, sewage-treating agent, but find that it is more sticky sometimes in fermentation medium optimization process, it the analysis found that Its thick substances is mainly polysaccharide material, the analysis found that its main polysaccharide material for galactolipin, glucose, rhamnose, sweet Dew sugar and glucuronic acid are formed；And test its highest polysaccharide material by different culture mediums and can reach more than 10g/L（Afterwards Face the present inventor team will have research report or patent is further disclosed）But if polysaccharide material is higher, it is remaining Content of reducing sugar is also higher, is not suitable for the preservation of later product, last we determine its training disclosed by the invention through overtesting Support base and culture process is proper, not only residual reduced sugar is less, and polysaccharide material is also that can play obvious effect, there is document Report polysaccharide can increase aspermia or oligospermia model mice androgone number and sperm concentration, its mechanism may suppress albumen with upregulation of apoptosis Bcl-w expression, downward bax expression are related, therefore the present inventor team has also carried out correlation test.
The bacillus amyloliquefaciens contained in the present invention, it is a kind of production gemma, the gram-positive bacteria of facultative aerobic, is had There is the features such as resistance is strong, antimicrobial spectrum is wide, metabolite is abundant, as probiotics, pathogen adheres to, biology is taken by force by suppressing Oxygen, antagonism pathogen, a variety of digestive ferments of secretion etc., build healthy intestinal environment, promote absorption of nutrient ingredients to utilize, improve livestock and poultry The general level of the health.The bacillus amyloliquefaciens YHSH-58 of the present invention is micro- in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China The common micro-organisms center preservation of biological inoculum preservation administration committee, deposit number are that CGMCC NO.13664 preservation dates are On 2 16th, 2017；It is that we separate in the intestinal contents of the wild wild boar in Hunan Province Longhui County beachhead town field Arrive, a variety of extracellular active materials, including lipopeptid class, peptides, protide etc. can be produced.These metabolites can suppress cause of disease Bacterium, virus etc..This bacillus amyloliquefaciens can produce Antagonistic protein TasA, with urfactin class antibacterial lipopeptids, can change Permeability of cell membrane, destroys membrane structure, to multi-drug resistant Escherichia coli, pseudomonas aeruginosa, enterococcus faecalis, cloaca intestines Bacillus, Pseudomonas aeruginosa, proteus, staphylococcus aureus have obvious inhibitory action.
The coronoid process dissipate capsule bacterium (Eurotium cristatum, EC) contained in the present invention is that gold is produced in black tea production process Colored dominant bacteria, is the principal element to form black tea distinguishing character.The present invention utilizes the coronoid process for isolating and purifying out in melanism tea Bulk bacteria carry out deep fermentation, ferment in different culture media and produce exocellular polysaccharide, although its polyoses content is relatively low, compared with peach Polysaccharide material caused by red pod sulphur bacterium has obvious difference, its mainly by rhamnose, arabinose, mannose, glucose, The monose such as galactolipin form, and the close weave under liquid fermentation condition of the coronoid process dissipate capsule bacterium in the present invention is in mycelia Ball, and constantly increase, the most of aging self-dissolving of fermentation later stage appearance, and itself contained substantial amounts of amino acid and unknown nutrition The factor discharges immediately, there is obvious beneficial effect to human body.
Beneficial effects of the present invention：
1st, complex microorganism preparations of the invention can increase aspermia or oligospermia model mice androgone number and sperm concentration, adjust and improve Mice serum sex hormone level；Its mechanism may be related with upregulation of apoptosis suppression albumen bcl-w expression, downward bax expression.
2. the complex microorganism preparations of the present invention contain substantial amounts of microbial polysaccharide material and active probiotic, there is soothing the liver tune Gas, kidney tonifying lead to essence, adjust the effect such as enteron aisle, can be obviously improved clinical symptoms and sign, improve Sperm Parameters and endocrine water It is flat, improve infertility and disease, the course of disease can be shortened, improve curative effect.
3rd, the few weak sperm sterility significant effect of complex microorganism preparations of the invention treatment, clinical practice does not find any Toxic side effect, has the advantages that efficient, safe, inexpensive, easy.Identical open research report is had no at present, illustrates the method It is a kind of effective new method of the few weak sperm sterility for the treatment of, is worthy of popularization.
4th, after complex micro organism fungicide of the invention enters enteron aisle with viable bacteria, to pathogenic bacteria such as staphylococcus, Escherichia coli There is antagonism, and have promotion growth to Bifidobacterium, Bacillus acidi lactici, bacteroid, peptostreptococcus, so that adjustable bacterium Group's imbalance.The complex micro organism fungicide of the present invention can promote body to produce antibacterial substance, kill pathogenic bacteria.In addition by taking by force Oxygen biological effect makes enteron aisle anoxic, is conducive to a large amount of anaerobic bacteria growths.
Embodiment
The separation and identification of 1 bacillus amyloliquefaciens of embodiment
In December, 2014, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain is in Hunan Province Longhui County It is isolated in the intestinal contents of the wild wild boar in beachhead town field.Specific method is:Take the enteron aisle content of wild wild boar Thing is added in the triangular flask for filling 10mL aqua sterilisas, and shake culture 30min (200r/min), then takes suspension 1mL to be placed in In 9mL sterile waters, 10 are made-2Diluted suspension liquid, and so on, it is made 10-5, 10-6Dilute suspension;It is each wherein to draw 100 U L, are added on nutrient broth medium tablet, uniform with coating rod coating, and each processing is repeated 3 times.By above-mentioned culture dish, put 1-2d is cultivated in 37 DEG C of constant incubators.Picking individual colonies are transferred to nutrient broth tablet culture, after growing bacterium colony, with inoculation Ring carries out line and isolates and purifies, and purifying bacterial strain is in 4 DEG C of preservations, numbering YHSH-58.
Monoclonal grows the bacterium colony to be formed to the bacterial strain that separation, purifying obtain on nutrient broth medium is circular, Surrounding is smooth, milky；Cell shape is rod-shaped, and cell dia is more than lum；Gemma is formed, does not form parasporal crystal;Aerobe
It is long;Catalase, oxydase reaction are the positive;Arginine dihydrolase, lysine take off shuttle enzyme, ornithine takes off shuttle enzyme, arteries and veins enzyme, Color
Propylhomoserin takes off shuttle enzyme reaction for feminine gender；VP is tested and nitrate reduction is the positive;Glucose, xylose, L- Arab can be utilized Sugar,
Mannose, lactose, sucrose, sorbose, semen armeniacae amarae former times;Melibiose, rhamnose, inositol etc. cannot be utilized.Optimal pH is 6- 8.5, optimum growth temperature is 25-38 DEG C, and bacillus amyloliquefaciens are accredited as by 16s rDNA.
2 bacillus amyloliquefaciens YHSH-58 bacteriostatic activity Preliminary Determinations of embodiment
Using the bacteriostatic activity of disc diffusion method measure bacillus amyloliquefaciens zymotic fluid.Use and connect with rubbing method sterile working Kind, every kind of bacterium connects 5 culture dishes, applied with spatula per 0.2 mL of ware inoculum concentration, takes appropriate medicinal extract heating water bath（Temperature is not More than 45 DEG C）Dissolving, then takes sterilizing filter paper disk (true 9 millimeters of footpath) to immerse each sample respectively and takes out for several minutes, is put after slightly dry Enter to be connected to the culture dish of strain, per 3, ware, each paper distance between commutator segments is equal, places in equilateral triangle, and each processing does 4 A repetition.Sterile water is immersed with filter paper to compare, be positioned at 28 DEG C at the same time（Bacteria Culture 24h, fungal culture 72h）, Antibacterial circle diameter is measured with crossing method, takes its average value as experimental result.Compared with distilled water.
Minimum inhibitory concentration（MIC）Measure
MIC is measured using doubling dilution, i.e., each sample is made into the liquid of every mL fermentation of bacillus liquid containing 10mg, Liquid is diluted to the dilution of 10~0.002mgmL-1 successively by coubling dilution again.Take tissue culturing plate（96 holes）, 190 μ L and 10 μ L bacteria suspensions are added per hole, while does and dilutes and sterile water control.Culture plate condition of culture is same as above, asepsis growth That hole liquid be minimum inhibitory concentration.Result of the test is shown in Table 1
1 bacillus amyloliquefaciens YHSH-58 bacteriostatic activity results of table
Escherichia coli Pseudomonas aeruginosa Enterococcus faecalis Enterobacter cloacae Pseudomonas aeruginosa Proteus Staphylococcus aureus
Bacteriostatic diameter (mm) 21.6 19.2 18.9 20.1 22.4 21.0 24.2
MIC 0.005 0.01 0.02 0.02 0.01 0.005 0.001
This bacillus amyloliquefaciens can produce Antagonistic protein as can be seen from Table 1, and class antibacterial lipopeptid, can change cell membrane and lead to Permeability, destroys membrane structure, green to multi-drug resistant Escherichia coli, pseudomonas aeruginosa, enterococcus faecalis, enterobacter cloacae Purulence bacillus, proteus, staphylococcus aureus have obvious inhibitory action.
The preparation of 3 complex microorganism preparations of embodiment
A kind of complex microorganism preparations for preventing aspermia or oligospermia, the raw material for preparing the microorganism formulation are by weight：Pink pod sulphur 35 parts of fermented liquid, 8 parts of coronoid process dissipate capsule bacterium seed, 40 parts of bacillus amyloliquefaciens zymotic fluid, 5 parts of brown sugar, soybean protein isolate 3 parts of powder, 2 parts of honey, 0.2 part of sodium chloride, 1 part of xylo-oligosaccharide, 80 parts of deionized water.
Wherein, the preparation method of the complex microorganism preparations of the prevention aspermia or oligospermia, includes the following steps：
Addition, 5 parts of brown sugar, 3 parts of soybean separation protein white powder, 2 parts of honey, 0.2 part of sodium chloride, xylo-oligosaccharide 1 in fermentation tank Part, 80 parts of deionized water, keep the temperature 50 minutes at 105 DEG C again after mixing, are then cooled to 30 DEG C；
Added in (1) coolant that step is produced：The 8 parts of 28-30 DEG C of ventilation cultures of coronoid process dissipate capsule bacterium seed, liquid-gas ratio per minute Throughput is 1:1, open stirring 150r/min culture 84 it is small when, detection thalline content be 45g/L, exocellular polysaccharide content is 1.2 g/ L, i.e., be transferred to pink 35 parts of pod sulphur fermented liquid to fermentation tank, continue culture 60 it is small when, it is 0.8g/L to detect its residual reduced sugar；Knot Beam second stage is fermented；
40 parts of bacillus amyloliquefaciens zymotic fluid is added in (2) zymotic fluid that step is cultivated, detect its total viable bacteria for 5.2 × 109CFU/ml, spore content are 3.8 × 109CFU/ml, you can filling, packed products.
Wherein, pink pod sulphur fermented liquid preparation method, includes the following steps：
, semi-solid seed activation culture：Pink pod sulphur strain is punctured in semi-solid purple sulphur photosynthetic bacteria culture medium, 25- 35 DEG C of illumination cultivations 7-10 days, bacterium line to be punctured redden and grow lawn, you can for the strain of activation
, seed culture：The strain of activation is seeded in seed fluid nutrient mediums of saccharomycete, 25-35 DEG C of temperature, intensity of illumination is： 1000-3000lux, illumination Anaerobic culturel 7-10 days detect OD650 >=1.2 of seed, and the cfu/ml of viable count >=600,000,000 is kind Sub- nutrient solution；
, fermented and cultured：By seed culture fluid and pink pod sulphur bacteria fermentation culture medium with 1：5 inoculum concentration inoculation, is trained in illumination Supporting Anaerobic culturel 7-10 days, 25-35 DEG C of cultivation temperature, intensity of illumination in tank is：1000-4000lux, mixing speed for 50 turns/ Minute, its OD650 >=4 to be detected, the cfu/ml of viable count >=1,000,000,000, polyoses content reaches 5 g/L, is pink pod sulphur bacterium fermentation Liquid；
Wherein, the purple sulphur photosynthetic bacteria culture medium of the semisolid is：Ammonium chloride 0.6g/L, potassium dihydrogen phosphate 0.6g/L, two water Calcium chloride 0.08g/L, magnesium chloride 0.3g/L, fructose 2g/L, 2 g/L of sodium acetate, natrium malicum 1g/L, nine water vulcanized sodium 0.2g/ L, agar 10g/L, 121 DEG C sterilize 15 minutes, with acetic acid tune PH to 7.0-7.2, wherein nine water vulcanized sodium are first configured to 0.1g/mL Individually sterilizing；
Wherein, the seed fluid nutrient mediums of saccharomycete is：Ammonium chloride 0.6g/L, potassium dihydrogen phosphate 0.8g/L, calcium chloride dihydrate 0.05g/L, magnesium chloride 0.3g/L, fructose 2g/L, 2 g/L of sodium acetate, glycerine 0.5g/L, nine water vulcanized sodium 0.2g/L, 121 DEG C go out Bacterium 15 minutes, with acetic acid tune PH to 7.0-7.2, individually sterilizes wherein nine water vulcanized sodium are first configured to 0.1g/mL；
Wherein, the pink pod sulphur bacteria fermentation culture medium is：Soybean protein isolate 20 g/L, ammonium chloride 0.6g/L, di(2-ethylhexyl)phosphate Hydrogen potassium 0.6g/L, calcium chloride dihydrate 0.08g/L, magnesium chloride 0.2g/L, fructose 20g/L, sodium acetate 2 g/L, honey 10g/L, sulphur Sodium thiosulfate 1g/L, 121 DEG C sterilize 15 minutes, with acetic acid tune PH to 7.0-7.2；
Wherein, pink pod sulphur bacterium is deposited in China General Microbiological Bacterial Strains Managing center by the present inventor, and numbering is CGMCC10344.Preservation date is on January 12nd, 2015.
Wherein, the preparation method of bacillus amyloliquefaciens zymotic fluid, includes the following steps：First, culture presevation pipe is taken out, is used LB solid mediums draw tablet and recover, when 33 DEG C of cultures 48 are small.Picking single bacterium colony is inoculated with 50 milliliters of LB trainings under tablet Support in base, when 33 DEG C of shake cultures 24 are small in the incubator.Seed uses 5% inoculum concentration, is seeded to liquid amount and is trained for 2L LB In the big triangular flasks of 5L for supporting base, when 33 DEG C of shake culture 20-28 are small, its bacterial concentration is detected, viable bacteria content is more than 2,000,000,000/milli Rise, you can as bacillus amyloliquefaciens liquid spawn；
2nd, using the bacillus amyloliquefaciens liquid spawn of step 1 culture as seed, 600L fermentation mediums is seeded to, are inoculated with Measure as 10%, open stirring 120r/min, minute ventilation volume liquid-gas ratio 1：When 0.8,33 DEG C of culture 24-36 is small, treat spore content not Less than 10,000,000,000/milliliter, you can terminate fermentation；
Wherein, the LB culture mediums：Peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, in solid medium Add agar 2%；
Wherein, the fermentation medium：Soybean protein isolate 40 g/L, glucose 10g/L, 20 g/L of soluble starch, albumen 8 g/L of peptone, 0.3 g/L of magnesium sulfate, 0.2 g/L of manganese sulfate, 0.5 g/L of sodium dihydrogen phosphate, 2.3 g/L of disodium hydrogen phosphate, pH7.2；The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes；
Wherein, the bacillus amyloliquefaciens are bacillus amyloliquefaciens YHSH-58, by the patentee in court of Beijing The China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation of the positive institute 3 of area's North Star West Road 1, deposit number are CGMCC NO.13664 preservation dates are on 2 16th, 2017.
Wherein, the coronoid process dissipate capsule bacterium seed liquor is prepared as follows：Coronoid process dissipate capsule bacterium culture presevation pipe is taken out, is used PDA solid mediums draw tablet and recover, and 28 DEG C are cultivated 5 days, and the streak inoculation of picking single bacterium colony is equipped with 150 millis under tablet In the eggplant bottle for rising PDA solid mediums, cultivate 5-7 days for 28 DEG C in the incubator, treat that lawn covers with eggplant bottle, produce a large amount of spores The i.e. 500 milliliters available sterile saline elution of son, adjusting spore concentration is 100,000,000 cfu/ml, is coronoid process dissipate capsule bacterium seed Liquid；
Wherein, coronoid process dissipate capsule bacterium strain is available from China General Microbiological culture presevation administrative center, numbering CGMCC 3.7928。
4 complex microorganism preparations of embodiment are in treatment mouse sterility result of the test
1 experiment packet and medication
There is small arrive to number greatly according to weight 40 male mices, number from 00 to 40, then 4 are randomly divided into random digits table Group：Blank group, model group, positive controls, test group.Blank group gives intraperitoneal injection of saline 2ml/d, other each groups are adopted Aspermia or oligospermia model is made with 0. 1% endoxan 2mg/d of intraperitoneal injection, the continuous 5d that injects, blank group and model group are given from the 6th day Lml/d physiological saline gavages are given, positive controls are given 150 μ of citric acid Clomifene aqueous solution of 0.2%, 2mg/ml by weight g.g-1.d-1Gavage is treated, and the complex microorganism preparations 0.15m1/20g gavages treatment of the production of embodiment 3 is given once daily in test group Gavage is treated, successive administration 35d.
2 result of the tests：
Each group serum T, the comparison of LH, FSH level
The results show model group serum FSH, LH levels are compared with other groups significantly rise (P<0. 01), and T levels are aobvious compared with other groups Writing reduces (P<0. 01)；Serum FSH between blank group, positive controls and test group, LH, T level differences are anticipated without statistics Justice (P>0. 05), is shown in Table 2.Show that the complex microorganism preparations of the present invention can be adjusted and improve mice serum sex hormone level.
2 each group serum T of table, the comparison of LH, FSH level
Each group sperm concentration result of variations
Model group sperm concentration is substantially less than blank group, shows that endoxan has the reproductive function of mouse more apparent influence, and Test group is contrasted compared with model group, and sperm concentration, which has, significantly rises (P<0. 0l), compared with positive controls, each treatment group's sperm Density has obvious rising (P<0.01), with the not statistically significant (P of blank group comparing difference>0. 05), is shown in Table 3.Show test group Aspermia or oligospermia can be preferably treated, increases mouse sperm quantity, and therapeutic effect is better than citric acid Clomifene.And safe and reliable
3 each group sperm concentration result of variations of table
5 complex microorganism preparations of embodiment are in treatment sterility result of the test
1 method：150 few weak sperm patients with infertility, are randomly divided into 2 groups, complex microorganism preparations are taken by treatment group 100 50ml/ times, three times per day, control group 50 takes wuzi yanzong pills, 9 grams every time, first, three times, continuously taking three months.Clothes Other medicines are refused to obey during medicine, avoiding eating or eating pungent, peppery, wine etc. less stimulates food and greasy food.Phase is carried out after 1 course for the treatment of of treatment Close inspection and statistics.Two groups of clinical efficacy is observed, the liter essence effect after treating 3 months and the shadow to sex hormone level Ring.
2nd, general information
2.1 packet：This group observation object is andrology's consulting patients in out-patient department, accepts infertile patients 150 for medical treatment altogether by above-mentioned standard, adopts It is divided into 2 groups with digital table method：Microbial bacterial agent treatment group 100, wuzi yanzong pills control group 50.
2.2 ages and the course of disease：The oldest 40 years old in treatment group 100, it is 22 years old, 22--29 Sui 48,30-35 Sui minimum 38, more than 35 years old 14, average 29 scholar 2.1 years old；The course of disease is most 10 years long, most 2 years short, average 5 scholar 1.4 years；Control group 50 In the oldest 40 years old, it is 23 years old minimum, 23--29 Sui 27,30-35 Sui 18, more than 35 years old 5, be averaged 28 ± 1.6 years； The course of disease is most 9 years long, most 2 years short, 4 ± 1.6 years average.
2.3 simultaneous diseases：Have prostatitis 61 concurrently, nonliquefaction of semen 53, Positive Anti-sperm Antibody 32, slight spermatic cord is quiet Arteries and veins varicose 19.
2.4 severity Scaling：Sperm concentration in treatment group 100>20 x 106/ m1 12, slight oligospermatism 39, Moderate 35, severe 14；Normal 10 of motility of sperm, slight asthénospermie 38, moderate 39, severe 13；Survive Rate is 44 of 41-60%, and survival rate is 46 of 21-40%, survival rate<10 of 20%；Aspermia or oligospermia 13, azoospermia 14, Aspermia or oligospermia+azoospermia 73.Sperm concentration in control group 50>20 × 106,/m1 10, slight oligospermatism 20, moderate 17, severe 3；Motility of sperm normal in 7 cases, slight asthénospermie 23, moderate 16, severe 4；Survival rate is 41- 60%22,21-40% 15,<20%3；Aspermia or oligospermia 7, azoospermia 9, aspermia or oligospermia+azoospermia 34.
3 criterions of therapeutical effect
According to WHO《The inspection of infertile couples standard and diagnosis handbook)) the effect of standard evaluated：
Clinical cure：The wife's side is pregnant；
It is effective：Sperm concentration after aspermia or oligospermia treatment>Ten b grades of 20 × 106/ml, azoospermia motility of sperm a level>50% or a grades> 25%, when survival rate 1 is small>60%；
Effectively：The lifting of sperm concentration after aspermia or oligospermia treatment>30%, the lifting of a+b grades or a grades of sperm motility after azoospermia treatment> 30%, lifting when survival rate 1 is small>30%0
It is invalid：The lifting of sperm concentration, survival rate and vigor after treatment<It is unchanged after 30%, or treatment.
4 treatment results
The inspection of pretherapy and post-treatment hepatic and renal function is no abnormal.Its clinical total effects is shown in Table 4, and treatment group, the clinic of control group are controlled More rate is respectively 45%, 11%, and total effective rate is respectively 99 %, 66% (P<0.01), two groups of curative effects relatively have significant difference (P<0.05 ).Sperm Parameters are relatively shown in Table 5, and treatment group, control group sperm concentration, sperm survival rate and sperm motility are put down and changed Kind to compare, difference has very significant (p<0.01 ).Two groups of prostatitis, nonliquefaction of semen, Positive Anti-sperm Antibody and spermatic cord Varication etc. and disease improve, and difference has very significant (p<0.01 ).Treatment group continued to take again again later with control group One course for the treatment of, its clinical cure rate can reach 87%, 35%；Significant difference.
Curative effect compares (n, %) after 4 two groups of treatments of table
Group Number of cases Clinical cure It is effective Effectively It is invalid Total effective rate（%）
Treatment group 100 45 48 6 1 99
Control group 50 11 12 10 17 66
5 Sperm Parameters of table compare
Conclusion：1 complex microorganism preparations can increase aspermia or oligospermia model mice androgone number and sperm concentration, its mechanism may be with Upregulation of apoptosis suppression albumen bcl-w expression, downward bax expression are related.
2. complex microorganism preparations contain substantial amounts of microbial polysaccharide material and active probiotic, there is soothing the liver regulating the qi flowing in the channels, kidney tonifying to lead to Essence, adjusts the effect such as enteron aisle, can be obviously improved clinical symptoms and sign, improve Sperm Parameters and Endocrine Levels, improve not Simultaneous disease is educated, the course of disease can be shortened, improves curative effect.
3rd, the few weak sperm sterility significant effect of complex microorganism preparations side's treatment, clinical practice do not find that any poison is secondary Effect, has the advantages that efficient, safe, inexpensive, easy.Identical open research report is had no at present, and it is to control to illustrate the method A kind of effective new method of few weak sperm sterility is treated, is worthy of popularization.

Claims (5)

1. a kind of complex microorganism preparations for preventing aspermia or oligospermia, it is characterised in that prepare the raw material of the microorganism formulation by weight Part it is：Pink 20-40 parts of pod sulphur fermented liquid, 5-10 parts of coronoid process dissipate capsule bacterium seed, bacillus amyloliquefaciens zymotic fluid 20-40 Part, 3-6 parts of brown sugar, 2-4 parts of soybean separation protein white powder, 1-2 parts of honey, 0.2-0.4 parts of sodium chloride, 0.5-2 parts of xylo-oligosaccharide, go 80 parts of ionized water.
2. the preparation method of the complex microorganism preparations of prevention aspermia or oligospermia according to claim 1, it is characterised in that including Following steps：
3-6 parts of brown sugar, 2-4 parts of soybean separation protein white powder, 1-2 parts of honey, 0.2-0.4 parts of sodium chloride are added in fermentation tank, it is low 0.5-2 parts of xylan, 80 parts of deionized water, keep the temperature 50 minutes at 105 DEG C again after mixing, are then cooled to 30 DEG C；
Added in (1) coolant that step is produced：The 5-10 parts of 28-30 DEG C of ventilation cultures of coronoid process dissipate capsule bacterium seed, liquid-gas ratio per minute Throughput is 1:1, open stirring 150r/min culture 72-96 it is small when, treat that thalline content reaches 40g/L, exocellular polysaccharide content reaches 1 G/L, i.e., be transferred to pink 20-40 parts of pod sulphur fermented liquid to fermentation tank, continue cultivate 48-72 it is small when, it is small to detect its residual reduced sugar In 1g/L；It can terminate second stage fermentation；
20-40 parts of bacillus amyloliquefaciens zymotic fluid is added in (2) zymotic fluid that step is cultivated, detects its total viable bacteria not less than 3 ×109CFU/ml, spore content are not less than 2 × 109CFU/ml, you can filling, packed products.
A kind of 3. complex microorganism preparations for preventing aspermia or oligospermia according to claim 1, it is characterised in that pink pod sulphur bacterium Zymotic fluid preparation method, includes the following steps：
, semi-solid seed activation culture：Pink pod sulphur strain is punctured in semi-solid purple sulphur photosynthetic bacteria culture medium, 25- 35 DEG C of illumination cultivations 7-10 days, bacterium line to be punctured redden and grow lawn, you can for the strain of activation
, seed culture：The strain of activation is seeded in seed fluid nutrient mediums of saccharomycete, 25-35 DEG C of temperature, intensity of illumination is： 1000-3000lux, illumination Anaerobic culturel 7-10 days detect OD650 >=1.2 of seed, and the cfu/ml of viable count >=600,000,000 is kind Sub- nutrient solution；
, fermented and cultured：By seed culture fluid and pink pod sulphur bacteria fermentation culture medium with 1：5 inoculum concentration inoculation, is trained in illumination Supporting Anaerobic culturel 7-10 days, 25-35 DEG C of cultivation temperature, intensity of illumination in tank is：1000-4000lux, mixing speed for 50 turns/ Minute, its OD650 >=4 to be detected, the cfu/ml of viable count >=1,000,000,000, polyoses content reaches 5 g/L, is pink pod sulphur bacterium fermentation Liquid；
Wherein, the purple sulphur photosynthetic bacteria culture medium of the semisolid is：Ammonium chloride 0.6g/L, potassium dihydrogen phosphate 0.6g/L, two water Calcium chloride 0.08g/L, magnesium chloride 0.3g/L, fructose 2g/L, 2 g/L of sodium acetate, natrium malicum 1g/L, nine water vulcanized sodium 0.2g/ L, agar 10g/L, 121 DEG C sterilize 15 minutes, with acetic acid tune PH to 7.0-7.2, wherein nine water vulcanized sodium are first configured to 0.1g/mL Individually sterilizing；
Wherein, the seed fluid nutrient mediums of saccharomycete is：Ammonium chloride 0.6g/L, potassium dihydrogen phosphate 0.8g/L, calcium chloride dihydrate 0.05g/L, magnesium chloride 0.3g/L, fructose 2g/L, 2 g/L of sodium acetate, glycerine 0.5g/L, nine water vulcanized sodium 0.2g/L, 121 DEG C go out Bacterium 15 minutes, with acetic acid tune PH to 7.0-7.2, individually sterilizes wherein nine water vulcanized sodium are first configured to 0.1g/mL；
Wherein, the pink pod sulphur bacteria fermentation culture medium is：Soybean protein isolate 20 g/L, ammonium chloride 0.6g/L, di(2-ethylhexyl)phosphate Hydrogen potassium 0.6g/L, calcium chloride dihydrate 0.08g/L, magnesium chloride 0.2g/L, fructose 20g/L, sodium acetate 2 g/L, honey 10g/L, sulphur Sodium thiosulfate 1g/L, 121 DEG C sterilize 15 minutes, with acetic acid tune PH to 7.0-7.2；
Wherein, pink pod sulphur bacterium is deposited in China General Microbiological Bacterial Strains Managing center by the present inventor, and numbering is CGMCC10344, preservation date are on January 12nd, 2015.
A kind of 4. complex microorganism preparations for preventing aspermia or oligospermia according to claim 1, it is characterised in that solution starch gemma The preparation method of bacillus fermentation liquid, includes the following steps：First, culture presevation pipe is taken out, drawing tablet with LB solid mediums carries out Recovery, when 33 DEG C of cultures 48 are small, picking single bacterium colony is inoculated with 50 milliliters of LB culture mediums under tablet, in the incubator 33 DEG C of shakes Swing culture 24 it is small when, seed use 5% inoculum concentration, be seeded to liquid amount be 2L LB culture mediums the big triangular flasks of 5L in, 33 DEG C When shake culture 20-28 is small, its bacterial concentration is detected, viable bacteria content is more than 2,000,000,000/milliliter, you can as bacillus amyloliquefaciens Liquid spawn；
2nd, using the bacillus amyloliquefaciens liquid spawn of step 1 culture as seed, 600L fermentation mediums is seeded to, are inoculated with Measure as 10%, open stirring 120r/min, minute ventilation volume liquid-gas ratio 1：When 0.8,33 DEG C of culture 24-36 is small, treat spore content not Less than 10,000,000,000/milliliter, you can terminate fermentation；
Wherein, the LB culture mediums：Peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, in solid medium Add agar 2%；
Wherein, the fermentation medium：Soybean protein isolate 40 g/L, glucose 10g/L, 20 g/L of soluble starch, albumen 8 g/L of peptone, 0.3 g/L of magnesium sulfate, 0.2 g/L of manganese sulfate, 0.5 g/L of sodium dihydrogen phosphate, 2.3 g/L of disodium hydrogen phosphate, pH7.2；The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes；
Wherein, the bacillus amyloliquefaciens are bacillus amyloliquefaciens YHSH-58, by the patentee in court of Beijing The China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation of the positive institute 3 of area's North Star West Road 1, deposit number are CGMCC NO.13664 preservation dates are on 2 16th, 2017.
A kind of 5. complex microorganism preparations for preventing aspermia or oligospermia according to claim 1, it is characterised in that the coronoid process Bulk bacteria seed liquor is prepared as follows：Coronoid process dissipate capsule bacterium culture presevation pipe is taken out, drawing tablet with PDA solid mediums carries out Recovery, 28 DEG C are cultivated 5 days, eggplant bottle of the picking single bacterium colony streak inoculation equipped with 150 milliliters of PDA solid mediums under tablet In, cultivate 5-7 days for 28 DEG C in the incubator, treat that lawn covers with eggplant bottle, produce the i.e. 500 milliliters available sterile life of a large amount of spores Salt water is managed, adjusting spore concentration is 100,000,000 cfu/ml, is coronoid process dissipate capsule bacterium seed liquor；
Wherein, coronoid process dissipate capsule bacterium strain is available from China General Microbiological culture presevation administrative center, numbering CGMCC 3.7928。
CN201711496530.XA 2017-12-31 2017-12-31 A kind of complex microorganism preparations for preventing aspermia or oligospermia and preparation method thereof Pending CN108030793A (en)

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Application publication date: 20180515